Multi-targeting short interfering RNAs

ABSTRACT

The present invention relates to novel short interfering RNA (siRNA) molecules that are multi-targeted. More specifically, the present invention relates to siRNA molecules that target two or more sequences. In one embodiment, multi-targeting siRNA molecules are designed to incorporate features of siRNA molecules and features of micro-RNA (miRNA) molecules. In another embodiment, multi-targeting siRNA molecules are designed so that each strand is directed to separate targets.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 13/287,493 filed 2 Nov. 2011 which is a division of U.S. patentapplication Ser. No. 12/021,604 filed 29 Jan. 2008, now U.S. Pat. No.8,071,752, which in turn is related to and claims priority under 35U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No.60/897,844 filed 29 Jan. 2007 and U.S. Provisional Patent ApplicationSer. No. 60/996,849 filed 7 Dec. 2007. Each application is incorporatedherein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

The present invention was made in part with Government support underGrant Numbers AI29329, AI42552 and HL07470 awarded by the NationalInstitutes of Health, Bethesda, Md. The Government has certain rights inthis invention.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled1954575SequenceListing.txt, created on 23 Sep. 2014, and is 11 kb insize. The information in the electronic format of the Sequence Listingis part of the present application and is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to novel short interfering RNA (siRNA)molecules that are multi-targeted. More specifically, the presentinvention relates to siRNA molecules that are capable target two or moresequences. In one embodiment, multi-targeting siRNA molecules aredesigned to incorporate features of siRNA molecules and features ofmicro-RNA (miRNA) molecules. In another embodiment, multi-targetingsiRNA molecules are designed so that each strand is directed to separatetargets.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

RNA interference (RNAi) is a process where double-stranded RNA triggerstogether with protein complexes downregulate mRNA complementary to thetriggers (Hannon and Rossi, 2004). The triggers are 19 nt long RNAduplexes with 2 nt 3′ overhangs and are referred to as short interferingRNAs (siRNAs). The protein complex, known as the RNA-induced silencingcomplex (RISC), incorporates one of the siRNA strands and RISC uses thisstrand as a template to recognize target mRNA. RISC then cleaves mRNAwith perfect or near-perfect complementarity to the guide strand. Shortinterfering RNAs are used as tools to downregulate specific genes andcan either give transient or—when stably integrated as short hairpinsRNAs (shRNAs) (Paddison et al., 2002)—stable suppression.

The siRNAs and shRNAs resemble intermediates in the processing pathwayof the endogenous microRNA (miRNA) genes (Bartel, 2004). Indeed, siRNAscan function as miRNAs and vice versa (Zeng et al., 2002; Doench et al.,2003). MicroRNAs, like siRNAs, use RISC to downregulate target genes,but unlike siRNAs, most animal miRNAs do not cleave the mRNA. Instead,miRNAs reduce protein output through translational suppression or polyAremoval and mRNA degradation (Wu et al., 2006). Known miRNA bindingsites are within mRNA 3′ UTRs; miRNAs seem to target sites withnear-perfect complementarity to nucleotides 2-8 from the miRNA's 5′ end(Rajewsky, 2006; Lim et al., 2005). This region is known as the seedregion. Because siRNAs and miRNAs are interchangeable, exogenous siRNAswill downregulate mRNAs with seed complementarity to the siRNA(Birmingham et al., 2006. Multiple target sites within a 3′ UTR givestronger downregulation (Doench et al., 2003).

Although many siRNA molecules have been developed for the treatment ofcancer disease, and other conditions, there remains a need for thedevelopment of additional new molecules and methods for the RNAitreatment of cancer, disease, and other conditions, particularly thedevelopment of new siRNA molecules.

SUMMARY OF THE INVENTION

The present invention relates to novel short interfering RNA (siRNA)molecules that are multi-targeted. More specifically, the presentinvention relates to siRNA molecules that target two or more sequences.In one embodiment, multi-targeting siRNA molecules are designed toincorporate features of siRNA molecules and features of micro-RNA(miRNA) molecules. In another embodiment, multi-targeting siRNAmolecules are designed so that each strand is directed to separatetargets.

In one aspect, the present invention provides a new approach fortargeting in which an siRNA can target multiple targets. As disclosedherein, this approach is termed “multi-targeting short interfering RNA”or “multi-targeting siRNA.” In one embodiment, a multi-targeting siRNAmolecule matches a desired coding region sequences, but also contain oneor more, preferably at least two, seed matches optimally spaced in the3′ UTR are developed. In this embodiment of the invention,multi-targeting siRNAs use multiple RNA interference (RNAi) pathways todown-regulate their intended target gene, thereby achieving more robustand potent gene down-regulation than tradition siRNAs have. TraditionalsiRNA are designed to use the cleavage pathway of RNAi only, but siRNAscan also induce miRNA-like translational suppression or polyAdegradation and cause transcriptional gene silencing (TGS). siRNAs thatcombine two or more of these pathways to down-regulate their intendedtarget(s) in accordance with the present invention give more robustdown-regulation than siRNAs that rely on only one of the pathways.

In a second embodiment, the present invention provides siRNA moleculesin which each strand of the molecule matches different desired codingregion sequences. In this embodiment, both strands of the siRNA moleculeare functional for the cleavage pathway of RNAi following cleavage byDicer.

In a second aspect, the present invention provides methods for designingmulti-targeting siRNAs of the first embodiment of the first aspect ofthe present invention. In one embodiment, multi-targeting siRNAscontaining siRNA and miRNA functions are designed using the followingprotocol.

1. Input one mRNA and one 3′ UTR target sequence.

2. Identify all 19mer siRNA candidates that have perfect complementarityto the mRNA.

3. For each siRNA candidate, identify miRNA-like target sites within the3′ UTR and remove candidates that have no sites.

4. Use an siRNA efficacy prediction algorithm to identify effectivecleavage target sites within the mRNA.

5. Order the siRNA candidates based on predicted miRNA-likedown-regulation. This prediction is based on the number of and distancebetween miRNA-like target sites within the 3′ UTR.

In a second embodiment, Step 3 is removed by using siRNAs that aremodified such that the guide strand is guaranteed to be preferentiallyloaded into RISC. Such modifications are well known to the skilledartisan.

In a third aspect, the present invention provides pharmaceuticalcompositions comprising an effective amount of a multi-targeting siRNAand a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides methods for treatinga broad spectrum of diseases and conditions, including, but not limitedto, cancer or cancerous disease, infectious disease, cardiovasculardisease, neurological disease, prion disease, inflammatory disease,autoimmune disease, pulmonary disease, renal disease, liver disease,mitochondrial disease, endocrine disease, reproduction related diseasesand conditions, and any other indications that can respond to the levelof an expressed gene product in a cell or organism. The term “infectiousagent” includes any virus (DNA or RNA virus), bacteria, fungus, orprotozoa which is capable of infection.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show multi-targeting siRNAs that combine cleavage andtranslational inhibition. FIG. 1A: A schematic of the multi-targetingsiRNA; red illustrates the siRNA “seed” region and green illustrates therest of the 19mer siRNA guide strand, i.e., the siRNA “cleavage”nucleotides. FIG. 1B: The multi-targeting siRNA can be designed totarget one transcript that has one cleavage site somewhere within themRNA and several seed sites within the 3′ UTR; red illustrates the siRNA“seed” region and green illustrates the rest of the 19mer siRNA guidestrand, i.e., the siRNA “cleavage” nucleotides. FIG. 1C: Alternatively,the multi-targeting siRNA can be designed to have a cleavage site withinone mRNA and several seed sites within the 3′ UTR of another transcript;red illustrates the siRNA “seed” region and green illustrates the restof the 19mer siRNA guide strand, i.e., the siRNA “cleavage” nucleotides.

FIG. 2 shows target sites for multi-targeting siRNAs against HIV pNL4-3.This figure shows a schematic of the pNL4-3 genome (coding regions, 5′and 3′ LTRs, and 3′ UTR) and the position of the target sites for thetwo multi-targeting siRNAs we have designed to target pNL4-3. The siRNAsare called “CU2” and “CU3”. CU2 has a cleavage site in GAG/POL and twoseed sites in the 3′ UTR; CU3 has a cleavage site in POL and three seedsites in the 3′ UTR.

FIG. 3 shows a schematic of the base-pairing between the CU2 and CU3siRNAs and their target sites in the pNL4-3 3′ UTR.

FIG. 4 shows seed sites for multi-targeting siRNAs against CCR5 and HIVpNL4-3. We have four siRNAs that have cleavage sites in CCR5 and seedsites in the pNL4-3 3′ UTR. The position of the seed-sites areillustrated in this figure.

FIG. 5 shows a schematic of the base-pairing between two of the foursiRNAs and their target sites in the pNL4-3 3′ UTR.

FIG. 6 shows a schematic of the base-pairing between two of the foursiRNAs and their target sites in the pNL4-3 3′ UTR.

FIG. 7 shows the effect of siRNAs on coding sequences of pNL4-3.

FIG. 8 shows the effect of miRNAs on 3′ UTRs of pNL4-3.

FIG. 9 shows the effect of siRNAs (C1-C4) on the CCR5 3′UTR and codingsequence regions.

FIG. 10 shows the effect of various miRNAs on the pNL4-3 3′ UTR region.

FIG. 11 shows the effect of various siRNAs on the pNL4-3 Luc reporterexpression.

FIG. 12 shows the analysis of bifunctional siRNA duplexes against Bcl6and STAT3 at the same time on a 15% native gel that was stained withSYBR Gold. Legend: 27=asymmetrical design, o=overhang, b=blunt, S=sensestrand only, AS=antisense strand only.

FIG. 13 shows the results of the psicheck assay for different designs ofmulti-targeting siRNAs for BS-1100.

FIG. 14 shows real-time PCR performed for STAT3 and Bcl6 mRNAs extractedfrom HEK293 cells 48 h after transfection of 50 nM siRNAs with RNAiMax.The first number after the target molecule name represents the targetsite in the mRNA sequence, the second number indicates asymmetricalDicer substrate (27mer) or the 21mer siRNA. STAT3 and BCL6 mRNA levelswere normalized to RPLP0 and are shown relative to the mock transfectedcontrol. Experiments were done in triplicate.

FIG. 15 shows real-time PCR performed for IFNβ, p56 and OAS1 mRNAsextracted from Raji cells 48 h after electroporation of 2 μg siRNAfollowing the Amaxa protocol. As positive control, Raji cells wereincubated with 1 U/μ1 IFNα for 4 h. Data was normalized to RPLP0 and isshown relative to the mock transfected control. Experiments were done induplicate.

FIGS. 16A and 16B show a proposed model for incorporation and processingof the Dicer-substrate multi-functional siRNAs. FIG. 16A: After bindingof the Dicer protein to the Dicer-substrate duplex (top center) theduplex is processed to siRNA and therefore the two strands of the duplexare incorporated into different RISCs. The lower left diagram depictsthe incorporation of the anti-CCR5 (red line) strand into RISC andcleavage of the target mRNA (green line). The lower right diagramdepicts incorporation of the U2 miRNA (black line) into RISC andtranslational inhibition of HIV proteins. FIG. 16B depicts the U2 andCCR5 strands against their targets. There are two seed sequences withoptimal distance (36 nt.) in the HIV (expressed from the pNL4-3 plasmid)3′ UTR that the U2 strand can bind to and exhibit miRNA activity. Thebottom strand of the multifunctional siRNA targets the CCR5 mRNA codingregion with perfect complementarity.

FIGS. 17A and 17B show the structures of U2 miRNA (left strands) boundto the two 3′ UTR sites of HIV (right strands). The U2 miRNA upstream ofthe seed sequence has differential complementarity against the HIV 3′UTR sites. FIG. 17A: Structure of the U2 miRNA bound to the first HIV 3′UTR site starting at nucleotide 362 of the 3′UTR. FIG. 17B: Structure ofthe U2 miRNA bound to the second HIV 3′ UTR site starting at nucleotide398 of the 3′UTR.

FIGS. 18A and 18B show a schematic of the reporter pU. FIG. 18A:Translational inhibition of the Renilla Luciferase gene by incorporationof the U2 miRNA into the RISC. FIG. 18B: The psiCheck reporter constructengineered to express the HIV 3′UTR downstream of the Renilla codingregion.

FIGS. 19A and 19B show a schematic of the reporter pCC. FIG. 19A: mRNAcleavage of the Renilla Luciferase gene by incorporation of anti-CCR5siRNA into the RISC. FIG. 19B: The psiCheck reporter constructengineered to express one kilo-bases of the CCR5 coding sequencedownstream of the Renilla coding region.

FIG. 20 shows the effect of the multi-functional siRNAs on the HIV-13′UTR and CCR5-CDS psiCheck Reporter Constructs. The syntheticDicer-substrate U2-CCR5-cds multi-functional siRNA (U2-C-C-D) wascotransfected with the psiCheck reporter expressing the HIV 3′ UTR (pU).Twenty four hours post transfection the cells were harvested, lysed andsubjected to Luciferase assay. The expression of Renilla Luciferase genewas reduced to 44.46 percent relative to the control irrelevant (pU-IRR)siRNA cotransfected with the pU reporter as well as the perfectlymatched U2 siRNA (top center of the chart) transfected with the pUreporter (pU-U2), 50.69 percent. The downregulation of the Renillaexpression is indicating processing of the Dicer-substratemultifunctional siRNA and incorporation of the U2 miRNA strand intoRISC. The efficiency and potency of the U2 miRNA in the context of themultifunctional Dicer-substrate duplex (left to right, first bar,44.46%)) was even better compared to the conventional 21 mer perfectsiRNA duplex of U2 (left to right, second bar, 50.69%). Theincorporation of the bottom strand of the Dicer-substratemultifunctional siRNA was validated by cotransfecting themulti-functional siRNA with the reporter psiCheck construct containingone kilobase pairs of the CCR5 coding region (pCC, target). Theexpression of the Renilla Luciferase gene was reduced to 37.71 percent(pCC-U2-C-C-D) (Left to right forth bar) relative to the irrelevantcontrol (pCC-IRR) (Left to right, last bar). The downregulation of theRenilla expression is indicating processing of the Dicer-substratemultifunctional siRNA and incorporation of the anti-CCR5 strand intoRISC. All of the transfections were done in triplicates.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel short interfering RNA (siRNA)molecules that are multi-targeted. A multi-targeting siRNA may bedirected against a single target gene or target sequence or may bedirected against multiple targets or target sequences. In oneembodiment, multi-targeting siRNA molecules are designed to incorporatefeatures of siRNA molecules and features of micro-RNA (miRNA) molecules.In another embodiment, multi-targeting siRNA molecules are designed sothat each strand is directed to separate targets.

The term “short interfering RNA” or “siRNA” as used herein refers to anynucleic acid molecule capable of inhibiting or down regulating geneexpression or viral replication, for example by mediating RNAinterference “RNAi” or gene silencing in a sequence-specific manner.

As used herein, the term “microRNA” or “miRNA” refers to any type ofinterfering RNA, including but not limited to, endogenous miRNA andartificial miRNA. Endogenous miRNA are small RNAs naturally present inthe genome which are capable of modulating the productive utilization ofmRNA. The term artificial miRNA includes any type of RNA sequence, otherthan endogenous miRNA, which is capable of modulating the productiveutilization of mRNA.

As used herein, “target gene” includes any nucleotide sequence, whichmay or may not contain identified gene(s), including, withoutlimitation, intergenic region(s), non-coding region(s), untranscribedregion(s), intron(s), exon(s), and transgene(s). The target gene can bea gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (i.e.,multi-target siRNA) of the invention. In one embodiment, inhibition,down-regulation or reduction with a multi-target siRNA molecule is belowthat level observed in the presence of an inactive or attenuatedmolecule. In another embodiment, inhibition, down-regulation, orreduction with multi-target siRNA molecules is below that level observedin the presence of, for example, a multi-target siRNA molecule withscrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence.

By “sense region” is meant a nucleotide sequence of a multi-target siRNAmolecule having complementarity to an antisense region of themulti-target siRNA molecule. In addition, the sense region of amulti-target siRNA molecule can comprise a nucleic acid sequence havinghomology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a multi-targetsiRNA molecule having complementarity to a target nucleic acid sequence.In addition, the antisense region of a multi-target siRNA molecule canoptionally comprise a nucleic acid sequence having complementarity to asense region of the multi-target siRNA molecule.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art. A percent complementarity indicatesthe percentage of contiguous residues in a nucleic acid molecule thatcan form hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%,70%, 80%, 90%, and 100% complementary). “Perfectly complementary” or“fully complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The functional definitions of miRNAs and siRNAs are based upon theirtarget site interactions and mechanism of action in altering geneexpression. miRNAs serve as guides for identifying short sequences (asfew as 7 bases), most often in the 3′ UTR to which they base pair andposit their associated argonaute and associated factors fortranslational inhibition. The base pairing region of miRNAs is calledthe “seed” sequence, usually consisting of bases 2-8 from the 5′ end ofthe antisense guide. Additional base pairing can take place, but onlythe seed sequence need pair to have functionality. siRNAs function viacomplete or near complete complementarity with their target sites, whichcan be anywhere on the message. They usually program Argonaute 2, whichcontains a cleavage domain, to cleave the target sequence between bases10 and 11 relative to the 5′ end of the antisense strand of the siRNA(reviewed in Filipowicz et al., 2005; Kim, 2005). The binding of miRNAsto the 3′ UTR can also trigger degradation, but this is not initiated bysite specific cleavage of the target, but appears to be a function ofthe inclusion of the miRNA/mRNA complex in processing bodies, orP-bodies (Liu et al., 2005).

In our studies of the genomic distribution of distances between pairs ofidentical miRNA seeds, we have found a propensity for moderate distancesgreater than 13 nucleotides between seed starts. Experimental datashowed that optimal downregulation is obtained when two seed sites areseparated by between 13 and 35 nucleotides, but can still be effectiveeven when they are 100 bases apart (Saetrom et al., 2007). Thesefindings are useful in developing improved miRNA target predictionalgorithms, as we have now incorporated the concept of sub-optimalversus optimal spacings between sites as a predictor of efficacy. Verypotent targets are likely to result in multiple miRNA-containingcomplexes binding within a narrowly defined region of the target tooptimize functional interaction. To illustrate, there are 12,735non-overlapping conserved pairs of hexamer seed sites throughout human3′ UTRs for the miRNAs in version 8.0 of miRBase (Griffiths-Jones etal., 2006), but only 2,257 pairs which are separated by more than 13 andless than 100 nucleotides. Our results also indicate that multipleco-expressed miRNAs will cooperate to down-regulate targets that containmultiple consecutive optimally spaced seed sites.

By analyzing the distance between seed sites of endogenous miRNAs andtransfected siRNAs, we also find that cooperative targeting of siteswith a separation in the optimal range can explain some of the siRNAoff-target effects that have been reported in the literature, sincesiRNAs can function as miRNAs when they have a seed match in the 3′ UTRof a non-targeted message (Jackson et al., 2006). This off-targeting maybe enhanced when they bind within the optimal distance from anendogenous miRNA. That is, our results indicate that siRNA off-targetingis related to cooperative downregulation by endogenous miRNAs.

Thus, and in accordance with one aspect of the present invention, a newapproach for targeting is provided in which an siRNA molecule targetsmultiple targets. As disclosed herein, this approach is termed“multi-targeting short interfering RNA” or “multi-targeting siRNA.” Inone embodiment, a multi-targeting siRNA molecule matches a desiredcoding region sequences, but also contain one or more, preferably atleast two, seed matches optimally spaced in the 3′ UTR are developed. Inthis embodiment of the invention, the multi-targeting siRNAs usemultiple RNA interference (RNAi) pathways to down-regulate theirintended target gene, thereby achieving more robust and potent genedown-regulation than tradition siRNAs have. Traditional siRNA aredesigned to use the cleavage pathway of RNAi only, but siRNAs can alsoinduce miRNA-like translational suppression or polyA degradation andcause transcriptional gene silencing (TGS). siRNAs that combine two ormore of these pathways to down-regulate their intended target(s) inaccordance with the present invention give more robust down-regulationthan siRNAs that rely on only one of the pathways.

This embodiment of the present invention currently focuses on combiningthe cleavage and miRNA-targeting pathways. We discovered that thedistance between multiple miRNA-like target sites dictates thedown-regulation, and we have used this result to create rules fordesigning siRNAs that give potent miRNA-like down-regulation combinedwith “traditional” mRNA cleavage. In theory, the approach can, however,also be combined with TGS to, for example, create siRNAs that cause TGS,mRNA cleavage, and miRNA-like down-regulation. Note that our “distancerules” also holds for target sites from different siRNAs, such that wecan use these rules to design different siRNAs that, if jointlyintroduced in the cell, cooperatively down-regulates the intendedtarget. This embodiment of the present invention adds another layer ofrobustness to our approach.

The present invention is not limited to target single genes. In someinstances, one can achieve more robust down-regulation of the intendedtarget by targeting additional genes, such as for example transcriptionfactors that positively regulate the intended target. It may not bepossible to find a cleavage site that is common for the two (or more)intended targets, but our approach gives the possibility to target onegene with one RNAi pathway (such as cleavage) and the other gene withanother pathway (such as miRNA-like targeting).

In summary, we know that siRNAs can use two mechanisms to target mRNAsfor downregulation. The cleavage mechanism is normally the desiredmechanism, whereas the “miRNA-like” downregulation is an undesirableoff-target effect. However, the present invention relates to the designof siRNAs that use both mechanisms to target specific mRNAs. Morespecifically, when designing siRNAs that target a specific mRNA, weensure that 1) the siRNA has a perfectly complementary target sitewithin the mRNA and 2) the siRNA has several miRNA-like target siteswithin the mRNA's 3′ UTR and that these target sites preferably arewithin the distance interval for optimal down-regulation.

In a second embodiment, multi-targeting siRNA molecules are provided inwhich each strand of the molecule matches different desired codingregion sequences. In this embodiment, both strands of the siRNA moleculeare functional for the cleavage pathway of RNAi following cleavage byDicer. In this embodiment, it is possible to target two different geneswith the cleavage RNAi pathway.

In one embodiment, multi-targeting siRNAs are designed using thefollowing protocol.

1. Input one mRNA and one 3′ UTR target sequence.

2. Identify all 19mer siRNA candidates that have perfect complementarityto the mRNA.

3. For each siRNA candidate, identify miRNA-like target sites within the3′ UTR and remove candidates that have no sites.

4. Use an siRNA efficacy prediction algorithm to identify effectivecleavage target sites within the mRNA.

5. Order the siRNA candidates based on predicted miRNA-likedown-regulation. This prediction is based on the number of and distancebetween miRNA-like target sites within the 3′ UTR.

In another embodiment, Step 3 is removed by using siRNAs that aremodified such that the guide strand is guaranteed to be preferentiallyloaded into RISC. Such modifications are well known to the skilledartisan.

In an additional embodiment, mRNA cleavage is incorporated intomulti-targeting siRNAs. For example, siRNAs have been designed whereboth strands have perfect complementarity to mRNA and can cause mRNAcleavage (Hossbach et al., 2006). This design can be incorporated in themulti-targeting siRNAs of the present invention such that both strandscan induce cleavage and have multiple miRNA-like target sites within thetarget 3′ UTR.

siRNA efficacy prediction algorithms are well known to the skilledartisan. Such algorithms include those described and referenced in Vertet al., 2006: Heale et al., 2005; Saetrom and Snove, 2004; Saetrom,2004; Chalk et al., 2004). Any suitable siRNA efficacy predictionalgorithm can be used in the present invention.

The siRNA molecules of the invention represent a novel therapeuticapproach to a broad spectrum of diseases and conditions, including, butnot limited to, cancer or cancerous disease, infectious disease,cardiovascular disease, neurological disease, prion disease,inflammatory disease, autoimmune disease, pulmonary disease, renaldisease, liver disease, mitochondrial disease, endocrine disease,reproduction related diseases and conditions, and any other indicationsthat can respond to the level of an expressed gene product in a cell ororganism. The term “infectious agent” includes any virus (DNA or RNAvirus), bacteria, fungus, or protozoa which is capable of infection.

In one embodiment, and for illustrative purposes only, multi-targetingsiRNAs to achieve robust down-regulation of HIV are prepared. We use two“multi-targeting” strategies to target HIV. In one embodiment, we havesiRNAs that have a cleavage site in a HIV coding region and miRNA-likesites in the 3′ HIV UTR. In a second embodiment, we have siRNAs thathave a cleavage site in CCR5—a co-receptor for HIV to enter hostcells—and miRNA-like sites in the 3′ HIV UTR. In the first embodiment,the miRNA-like target sites in the 3′ UTR ensures that any mRNA notcleaved by the siRNA instead gets down-regulated by the “miRNA”-pathway.It also gives robustness against escape mutants, as the virus must havemutations in both the cleavage and miRNA-like sites to escape siRNAdown-regulation and propagate. In the second embodiment, the cleavagesites in CCR5 prevent HIV integration and the miRNA-like sites in theHIV 3′ UTR serves as a backup to down-regulate any virus that shouldenter the cell.

As described above, the major objective in using this approach for HIVis to minimize viral escape mutants. Thus, if mutations arise in thecoding region, abrogating the effect of the siRNA, the siRNA can stillfunction as a miRNA by interacting with sites in the 3′ UTR suppressviral protein expression and even promote P-body associated degradationby this mechanism. Of course the same is true for a mutation in the 3′UTR, which may abrogate the potency of the miRNAs but the siRNA canstill function.

In another embodiment, and for illustrative purposes only,multi-targeting siRNAs to achieve robust down-regulation ofNon-Hodgkin's lymphomas (NHLs) are prepared. NHLs comprise a group ofheterogeneous lymphoid malignancies for which conventional chemo- andradiotherapy approaches are rarely curative and many lymphomas relapsewithin the first year. One hallmark of many types of B-cell lymphomas isthe constitutive expression of oncogenes such as the transcriptionfactors Bcl6, STAT3 and cMyc and the anti-apoptotic protein Bcl2. Overexpression of these genes causes uncontrolled proliferation, survival ofmalignant cells and protection against ionizing radiation and manycommonly used chemotherapeutics, making knockdown of these genes by RNAinterference (RNAi) a rational strategy for therapeutic intervention.RNAi is a conserved endogenous mechanism in which small interfering RNAs(siRNAs) suppress target-specific gene expression by promoting mRNAdegradation. We have designed potent Dicer-substrate siRNAs that showimproved efficacy at lower concentrations compared with conventional21mer siRNAs. In addition we have designed bifunctional siRNA duplexesthat provide two guide strands simultaneously suggesting the reductionof effective drug concentration, lower production costs, and decrease ofoff-target effects compared to conventional siRNAs.

In addition, the present invention provides a method for treatingdiseases. The molecules of the present invention are administered topatients in need of treatment using conventional pharmaceuticalpractices or as described herein. Suitable pharmaceutical practices aredescribed in Remington: The Science and Practice of Pharmacy, 21^(st)Ed., University of Sciences in Philadelphia, Ed., Philadelphia, 2005.

The siRNA molecule may have different forms, including a single strand,a paired double strand (dsRNA) or a hairpin (shRNA) and can be produced,for example, either synthetically or by expression in cells. In oneembodiment, DNA sequences for encoding the sense and antisense strandsof the siRNA molecule to be expressed directly in mammalian cells can beproduced by methods known in the art, including but not limited to,methods described in U.S. published application Nos. 2004/0171118 A1,2005/0244858 A1 and 2005/0277610 A1, each incorporated herein byreference. The siRNA molecules are coupled to carrier molecules, such asCpG oligodeoxynucleotides using the techniques described in U.S.provisional patent application Ser. No. 60/897,495 filed 26 Jan. 2007,U.S. patent application Ser. No. 11/966,423 filed 28 Dec. 2007 andInternational patent application No. PCT/US2007/026432 filed 28 Dec.2007, each incorporated herein by reference.

In one aspect, DNA sequences encoding a sense strand and an antisensestrand of a siRNA specific for a target sequence of a gene areintroduced into mammalian cells for expression. To target more than onesequence in the gene (such as different promoter region sequences and/orcoding region sequences), separate siRNA-encoding DNA sequences specificto each targeted gene sequence can be introduced simultaneously into thecell. In accordance with another embodiment, mammalian cells may beexposed to multiple siRNAs that target multiple sequences in the gene.

The siRNA molecules generally contain about 19 to about 30 base pairs,and preferably are designed to cause methylation of the targeted genesequence. In one embodiment, the siRNA molecules contain about 19-23base pairs, and preferably about 21 base pairs. In another embodiment,the siRNA molecules contain about 24-28 base pairs, and preferably about26 base pairs. In a further embodiment, the dsRNA has an asymmetricstructure, with the sense strand having a 25-base pair length, and theantisense strand having a 27-base pair length with a 2 base 3′-overhang.See, for example, U.S. published application Nos. 2005/0244858 A1 and2005/0277610 A1, each incorporated herein by reference. In anotherembodiment, this dsRNA having an asymmetric structure further contains 2deoxynucleotides at the 3′end of the sense strand in place of two of theribonucleotides. Individual siRNA molecules also may be in the form ofsingle strands, as well as paired double strands (“sense” and“antisense”) and may include secondary structure such as a hairpin loop.Individual siRNA molecules could also be delivered as precursormolecules, which are subsequently altered to give rise to activemolecules. Examples of siRNA molecules in the form of single strandsinclude a single stranded anti-sense siRNA against a non-transcribedregion of a DNA sequence (e.g. a promoter region).

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene.

The precursor RNAi molecule, may also have one or more of the followingadditional properties: (a) the antisense strand has a right shift fromthe typical 21mer and (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairings. A“typical” 21mer siRNA is designed using conventional techniques, such asdescribed above. This 21mer is then used to design a right shift toinclude 1-7 additional nucleotides on the 5′ end of the 21mer. Thesequence of these additional nucleotides may have any sequence. Althoughthe added ribonucleotides may be complementary to the target genesequence, full complementarity between the target sequence and the siRNAis not required. That is, the resultant siRNA is sufficientlycomplementary with the target sequence. The first and secondoligonucleotides are not required to be completely complementary. Theyonly need to be substantially complementary to anneal under biologicalconditions and to provide a substrate for Dicer that produces a siRNAsufficiently complementary to the target sequence. In one embodiment,the dsRNA has an asymmetric structure, with the antisense strand havinga 25-base pair length, and the sense strand having a 27-base pair lengthwith a 2 base 3′-overhang. In another embodiment, this dsRNA having anasymmetric structure further contains 2 deoxynucleotides at the 3′ endof the antisense strand.

Suitable dsRNA compositions that contain two separate oligonucleotidescan be linked by a third structure. The third structure will not blockDicer activity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene. In oneembodiment, the third structure may be a chemical linking group. Manysuitable chemical linking groups are known in the art and can be used.Alternatively, the third structure may be an oligonucleotide that linksthe two oligonucleotides of the dsRNA is a manner such that a hairpinstructure is produced upon annealing of the two oligonucleotides makingup the dsRNA composition. The hairpin structure will not block Diceractivity on the dsRNA and will not interfere with the directeddestruction of the RNA transcribed from the target gene.

The sense and antisense sequences may be attached by a loop sequence.The loop sequence may comprise any sequence or length that allowsexpression of a functional siRNA expression cassette in accordance withthe invention. In a preferred embodiment, the loop sequence containshigher amounts of uridines and guanines than other nucleotide bases. Thepreferred length of the loop sequence is about 4 to about 9 nucleotidebases, and most preferably about 8 or 9 nucleotide bases.

In another embodiment of the present invention, the dsRNA, i.e., theprecursor RNAi molecule, has several properties which enhances itsprocessing by Dicer. According to this embodiment, the dsRNA has alength sufficient such that it is processed by Dicer to produce an siRNAand at least one of the following properties: (i) the dsRNA isasymmetric, e.g., has a 3′ overhang on the sense strand and (ii) thedsRNA has a modified 3′ end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the longest strand in the dsRNAcomprises 24-30 nucleotides. In one embodiment, the sense strandcomprises 24-30 nucleotides and the antisense strand comprises 22-28nucleotides. Thus, the resulting dsRNA has an overhang on the 3′ end ofthe sense strand. The overhang is 1-3 nucleotides, such as 2nucleotides. The antisense strand may also have a 5′ phosphate.

Modifications can be included in the dsRNA, i.e., the precursor RNAimolecule, so long as the modification does not prevent the dsRNAcomposition from serving as a substrate for Dicer. In one embodiment,one or more modifications are made that enhance Dicer processing of thedsRNA. In a second embodiment, one or more modifications are made thatresult in more effective RNAi generation. In a third embodiment, one ormore modifications are made that support a greater RNAi effect. In afourth embodiment, one or more modifications are made that result ingreater potency per each dsRNA molecule to be delivered to the cell.Modifications can be incorporated in the 3′-terminal region, the5′-terminal region, in both the 3′-terminal and 5′-terminal region or insome instances in various positions within the sequence. With therestrictions noted above in mind any number and combination ofmodifications can be incorporated into the dsRNA. Where multiplemodifications are present, they may be the same or different.Modifications to bases, sugar moieties, the phosphate backbone, andtheir combinations are contemplated. Either 5′-terminus can bephosphorylated.

In another embodiment, the antisense strand is modified for Dicerprocessing by suitable modifiers located at the 3′ end of the antisensestrand, i.e., the dsRNA is designed to direct orientation of Dicerbinding and processing. Suitable modifiers include nucleotides such asdeoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and thelike and sterically hindered molecules, such as fluorescent moleculesand the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl groupfor the 2′-deoxyribofuranosyl sugar normally present in dNMPs. Othernucleotide modifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′ end of the antisensestrand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further invention, two terminal DNA bases arelocated on the 3′ end of the antisense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of thesense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of modifications contemplated for the sugar moietyinclude 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, anddeoxy modifications and the like (see, e.g., Amarzguioui et al., 2003).Examples of modifications contemplated for the base groups includeabasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like.Locked nucleic acids, or LNA's, could also be incorporated. Many othermodifications are known and can be used so long as the above criteriaare satisfied. Examples of modifications are also disclosed in U.S. Pat.Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patentapplication No. 2004/0203145 A1, each incorporated herein by reference.Other modifications are disclosed in Herdewijn (2000), Eckstein (2000),Rusckowski et al. (2000), Stein et al. (2001) and Vorobjev et al.(2001), each incorporated herein by reference.

Additionally, the siRNA structure can be optimized to ensure that theoligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention a 27-bpoligonucleotide of the dsRNA structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand.

RNA may be produced enzymatically or by partial/total organic synthesis,and modified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as described in Verma andEckstein (1998).

In another aspect, the present invention provides for a pharmaceuticalcomposition comprising the siRNA of the present invention. The siRNAsample can be suitably formulated and introduced into the environment ofthe cell by any means that allows for a sufficient portion of the sampleto enter the cell to induce gene silencing, if it is to occur. Manyformulations for dsRNA are known in the art and can be used so long assiRNA gains entry to the target cells so that it can act. See, e.g.,U.S. published patent application Nos. 2004/0203145 A1 and 2005/0054598A1, each incorporated herein by reference. For example, siRNA can beformulated in buffer solutions such as phosphate buffered salinesolutions, liposomes, micellar structures, and capsids. Formulations ofsiRNA with cationic lipids can be used to facilitate transfection of thedsRNA into cells. For example, cationic lipids, such as lipofectin (U.S.Pat. No. 5,705,188, incorporated herein by reference), cationic glycerolderivatives, and polycationic molecules, such as polylysine (publishedPCT International Application WO 97/30731, incorporated herein byreference), can be used. Suitable lipids include Oligofectamine,Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals,Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be usedaccording to the manufacturer's instructions.

It can be appreciated that the method of introducing siRNA into theenvironment of the cell will depend on the type of cell and the make upof its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the siRNA can be added directly to the liquidenvironment of the cells. Lipid formulations can also be administered toanimals such as by intravenous, intramuscular, or intraperitonealinjection, or orally or by inhalation or other methods as are known inthe art. When the formulation is suitable for administration intoanimals such as mammals and more specifically humans, the formulation isalso pharmaceutically acceptable. Pharmaceutically acceptableformulations for administering oligonucleotides are known and can beused. In some instances, it may be preferable to formulate siRNA in abuffer or saline solution and directly inject the formulated dsRNA intocells, as in studies with oocytes. The direct injection of dsRNAduplexes may also be done. For suitable methods of introducing siRNA seeU.S. published patent application No. 2004/0203145 A1, incorporatedherein by reference. In a further embodiment, the siRNA is delivered bya carrier molecule such as CpG oligodeoxynucleotides as described inprovisional patent application Ser. No. 60/897,495, filed 26 Jan. 2007,incorporated herein by reference.

Suitable amounts of siRNA must be introduced and these amounts can beempirically determined using standard methods. Typically, effectiveconcentrations of individual siRNA species in the environment of a cellwill be about 50 nanomolar or less 10 nanomolar or less, or compositionsin which concentrations of about 1 nanomolar or less can be used. Inother embodiment, methods utilize a concentration of about 200 picomolaror less and even a concentration of about 50 picomolar or less can beused in many circumstances.

The method can be carried out by addition of the siRNA compositions toany extracellular matrix in which cells can live provided that the siRNAcomposition is formulated so that a sufficient amount of the siRNA canenter the cell to exert its effect. For example, the method is amenablefor use with cells present in a liquid such as a liquid culture or cellgrowth media, in tissue explants, or in whole organisms, includinganimals, such as mammals and especially humans.

Expression of a target gene can be determined by any suitable method nowknown in the art or that is later developed. It can be appreciated thatthe method used to measure the expression of a target gene will dependupon the nature of the target gene. For example, when the target geneencodes a protein the term “expression” can refer to a protein ortranscript derived from the gene. In such instances the expression of atarget gene can be determined by measuring the amount of mRNAcorresponding to the target gene or by measuring the amount of thatprotein. Protein can be measured in protein assays such as by stainingor immunoblotting or, if the protein catalyzes a reaction that can bemeasured, by measuring reaction rates. All such methods are known in theart and can be used. Where the gene product is an RNA species expressioncan be measured by determining the amount of RNA corresponding to thegene product. The measurements can be made on cells, cell extracts,tissues, tissue extracts or any other suitable source material.

The determination of whether the expression of a target gene has beenreduced can be by any suitable method that can reliably detect changesin gene expression. Typically, the determination is made by introducinginto the environment of a cell undigested siRNA such that at least aportion of that siRNA enters the cytoplasm and then measuring theexpression of the target gene. The same measurement is made on identicaluntreated cells and the results obtained from each measurement arecompared.

The siRNA can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a siRNA andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a siRNAeffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of a RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

The phrase “pharmaceutically acceptable carrier” refers to a carrier forthe administration of a therapeutic agent. Exemplary carriers includesaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. The pharmaceutically acceptable carrier of thedisclosed dsRNA composition may be micellar structures, such as aliposomes, capsids, capsoids, polymeric nanocapsules, or polymericmicrocapsules.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound dsRNA into the cell. They include polymericand monomeric materials, especially including polybutylcyanoacrylate. Asummary of materials and fabrication methods has been published (seeKreuter, 1991). The polymeric materials which are formed from monomericand/or oligomeric precursors in the polymerization/nanoparticlegeneration step, are per se known from the prior art, as are themolecular weights and molecular weight distribution of the polymericmaterial which a person skilled in the field of manufacturingnanoparticles may suitably select in accordance with the usual skill.

Suitably formulated pharmaceutical compositions of this invention can beadministered by any means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general a suitable dosage unit of siRNA will be in the range of 0.001to 0.25 milligrams per kilogram body weight of the recipient per day, orin the range of 0.01 to 20 micrograms per kilogram body weight per day,or in the range of 0.01 to 10 micrograms per kilogram body weight perday, or in the range of 0.10 to 5 micrograms per kilogram body weightper day, or in the range of 0.1 to 2.5 micrograms per kilogram bodyweight per day. Pharmaceutical composition comprising the siRNA can beadministered once daily. However, the therapeutic agent may also bedosed in dosage units containing two, three, four, five, six or moresub-doses administered at appropriate intervals throughout the day. Inthat case, the siRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage unit. The dosage unitcan also be compounded for a single dose over several days, e.g., usinga conventional sustained release formulation which provides sustainedand consistent release of the siRNA over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.Regardless of the formulation, the pharmaceutical composition mustcontain siRNA in a quantity sufficient to inhibit expression of thetarget gene in the animal or human being treated. The composition can becompounded in such a way that the sum of the multiple units of siRNAtogether contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsRNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

In a further aspect, the present invention relates to a method for TGSin a mammalian, including human, cell. The method comprises introducingthe siRNA into the appropriate cell. The term “introducing” encompassesa variety of methods of introducing DNA into a cell, either in vitro orin vivo. Such methods include transformation, transduction,transfection, and infection. Vectors are useful and preferred agents forintroducing DNA encoding the siRNA molecules into cells. The introducingmay be accomplished using at least one vector. Possible vectors includeplasmid vectors and viral vectors. Viral vectors include retroviralvectors, lentiviral vectors, or other vectors such as adenoviral vectorsor adeno-associated vectors. In one embodiment, the DNA sequences areincluded in separate vectors, while in another embodiment, the DNAsequences are included in the same vector. The DNA sequences may beinserted into the same vector as a multiple cassettes unit. Alternatedelivery of siRNA molecules or DNA encoding siRNA molecules into cellsor tissues may also be used in the present invention, includingliposomes, chemical solvents, electroporation, viral vectors,pinocytosis, phagocytosis and other forms of spontaneous or inducedcellular uptake of exogenous material, as well as other delivery systemsknown in the art. In a further embodiment, the siRNA is delivered by acarrier molecule such as CpG oligodeoxynucleotides or RNA aptamers asdescribed in further detail herein.

Suitable promoters include those promoters that promote expression ofthe interfering RNA molecules once operatively associated or linked withsequences encoding the RNA molecules. Such promoters include cellularpromoters and viral promoters, as known in the art. In one embodiment,the promoter is an RNA Pol III promoter, which preferably is locatedimmediately upstream of the DNA sequences encoding the interfering RNAmolecule. Various viral promoters may be used, including, but notlimited to, the viral LTR, as well as adenovirus, SV40, and CMVpromoters, as known in the art.

In one embodiment, the invention uses a mammalian U6 RNA Pol IIIpromoter, and more preferably the human U6snRNA Pol III promoter, whichhas been used previously for expression of short, defined ribozymetranscripts in human cells (Bertrand et al., 1997; Good et al., 1997).The U6 Pol III promoter and its simple termination sequence (four to sixuridines) were found to express siRNAs in cells. Appropriately selectedinterfering RNA or siRNA encoding sequences can be inserted into atranscriptional cassette, providing an optimal system for testingendogenous expression and function of the RNA molecules.

In a further aspect, the invention provides a method for TGS in amammalian, including human, cell comprising introducing into the cellDNA sequences encoding a sense strand and an antisense strand of ansiRNA, which is specific for a target sequence in the gene to besilenced, preferably under conditions permitting expression of the siRNAin the cell, and wherein the siRNA directs methylation of said gene ofinterest. In an embodiment, methylation is directed to a sequence in thepromoter region of the gene. Alternately, methylation is directed to asequence in the coding region. Target sequences can be any sequence in agene that has the potential for methylation. In a preferred embodiment,the target sequences may contain CpG islands. The directed methylationcan lead to inactivation of the gene. To target more than one sequencein the gene (such as different promoter region sequences and/or codingregion sequences), separate siRNA-encoding DNA sequences specific toeach targeted gene sequence can be introduced simultaneously into thecell. In addition, cells may be exposed to multiple siRNAs that targetmultiple sequences in the gene.

Once a target sequence or sequences have been identified for methylationin accordance with the invention, the appropriate siRNA can be produced,for example, either synthetically or by expression in cells. In a oneembodiment, the DNA sequences encoding the sense and antisense strandsof the siRNA molecule can be generated by PCR. In another embodiment,the siRNA encoding DNA is cloned into a vector, such as a plasmid orviral vector, to facilitate transfer into mammals. In anotherembodiment, siRNA molecules may be synthesized using chemical orenzymatic means.

To facilitate nuclear retention and increase the level of methylation,the sense and antisense strands of the siRNA molecule may be expressedin a single stranded form, for example as a stem loop structure, asdescribed above. Alternatively, or in concomitance, the factor(s)involved in the active cellular transport of siRNA's, such as Exportin5, may be downregulated employing synthetic siRNA, antisense, ribozymes,or any other nucleic acid, antibody or drug, proven to be effective indownregulating the gene(s) of interest.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Russell, 1984, Molecular biology of plants: alaboratory course manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Anand, Techniques for the Analysis of ComplexGenomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide toYeast Genetics and Molecular Biology (Academic Press, New York, 1991);Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.); Nucleic Acid Hybridization (B. D. Hames & S.J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S.J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Fire et al.,RNA Interference Technology: From Basic Science to Drug Development,Cambridge University Press, Cambridge, 2005; Schepers, RNA Interferencein Practice, Wiley-VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts& Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference,Editing, and Modification: Methods and Protocols (Methods in MolecularBiology), Human Press, Totowa, N.J., 2004; Sohail, Gene Silencing by RNAInterference: Technology and Application, CRC, 2004; Clarke and Sanseau,microRNA: Biology, Function & Expression (Nuts & Bolts series), DNAPress, 2006.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 siRNAs Targeting HIV pNL4-3 Strain and CCR5 Gene

We have designed and tested multi-targeting siRNAs that target the HIVpNL4-3 strain (Table 1) and the CCR5 gene (Table 2), which is aco-receptor for HIV to enter host cells. All siRNAs have miRNA-liketarget sites within HIV 3′ UTR, but the cleavage target varies for thedifferent siRNAs. We designed the CCR5 siRNAs (Table 2) such that their3′ UTR sites have near-optimal distances to the HIV-targeting siRNAs(Table 1). These siRNAs give optimal cooperative miRNA-like targeting ofthe 3′ UTR when jointly introduced in the cell. The effects of thesemulti-targeting siRNAs on pNL4-3 are shown in FIGS. 7-11.

TABLE 1  siRNAs Designed Against pNL4-3 gag-pol and 3′ UTR SenseAntisense (SEQ ID NO:) (SEQ ID NO:) CDS 3′ UTR ID 5′-3′ 3′-5′ site sitesDist CU3 ucaggaaguaua aaugcaguauac 2132 219, 303, 84, cugcauu (1)uuccuga (2) 422 119 CU2 gagcuucagguu uuccccaaaccu 1384 362, 398 36uggggaa (3) gaagcuc (4) U2 uggacuuuugac uuccccagucaa None 362, 398 36uggggaa (5) aagucca (6) P2 uuccccauuuag uuccccacuaaa None 362, 398 36uggggaa (7) uggggaa (8)

-   -   The table lists the siRNA ID; the siRNA sense and antisense        sequences; the location of the cleavage site within pol-gag; the        location of the siRNAs' 3′ UTR seed sites; and the distance        between the seed sites. These sites are shown schematically in        FIG. 2 and the 3′ UTR target cites are shown in FIG. 3.

TABLE 2  siRNAs Designed Against CCR5 mRNA and pNL4-3 3′ UTRsense/antisense 3′ (SEQ ID NO:/SEQ ID NO:) CCR5 UTR Dist with Dist withID 5′-3′/3′-5′ site sites Dist CU3 CU2 CCR5_1 gagaggagucagagagaau/ 1034187, 55 32, 23, 55, 120, auucucucugacuccucuc (3′ UTR) 242 61, 119 36(9/10) CCR5_2 aguccaaucuaugacauca/ 18 330 84, 27, 32, 36ugaugucauagauuggacu (CDS) 92 (11/12) CCR5_3 ucugguuugcagagcuuga/ 1160198, 140 21, 84, 140, 24, ucaagcucugcaaaccaga (3′ UTR) 338 35, 84 36(13/14) CCR5_4 ggugucgaaaugagaagaa/ 667 24, 146 146, 49, 146, 192,uucuucucauuucgacacc (CDS) 170 84, 119 36 (15/16)

-   -   The table lists the siRNA ID; the siRNA sense and antisense        sequences; the location of the cleavage site within CCR5; the        location of the siRNAs' 3′ UTR seed sites; and the distance        between the seed sites; and the distances between the siRNA's        and the seed sites for siRNAs CU3 and CU2. These sites are shown        schematically in FIG. 4 and the 3′ UTR target cites are shown in        FIGS. 5 and 6.

Example 2 Multi-Targeting siRNAs

Other multi-targeting siRNAs have been developed to several combinationsof siRNA/miRNA targets for pNL4-3. These multi-targeting siRNAs areshown in Table 3.

TABLE 3  Multi-targeting siRNAs Designed Against TargetCoding Sequences and 3′ UTR in NL4-3 CDS siRNA (sense) (SEQ ID NO:) 3′UTR sites Dist env seq aagaguggugcagagagaa (17) 187, 240, 53 242 gag-polucaggaaguauacugcauu (18) 219, 303, 84, seq 422 119 gag seqgagcuucagguuuggggaa (19) 362, 398 36 Rev seq gcccgaaggaauagaagaa (20)24, 170 144 Vif seq acauauuggggucugcaua (21) 219, 303, 84, 422 119vpr seq ggaacaagccccagaagac (22) 24, 170 144

-   -   The table lists coding sequences in pNL4-3 (M19921) that are        targeted by at least one multi-targeting siRNA. The sequences        for tat and vpu did not have any potential siRNA/miRNA        combinations. The table gives an example of the siRNA; the        location of the siRNAs 3′ UTR seed sites; and the distance        between the seed sites.

Each of the sequences listed in Table 3 are tested using chemicallysynthesized siRNAs and co-transfection assays of the siRNAs with pNL4-3viral DNA. The ability of the siRNA to function is tested inco-transfections with pNL4-3 proviral DNA in 293 cells. Whether or notthe siRNAs are also functioning as miRNAs it tested by cloning the NL4-33′ UTR into a psiCHECK vector (Snove and Rossi, 2006) and monitoring theknockdown of luciferase expression in co-transfection assays.

Example 3 Multi-Targeting siRNAs to Two Targets

A hallmark of many types of B-cell lymphomas is the constitutiveexpression of oncogenes such as the transcription factors Bcl-6, STAT3and c-Myc and the anti-apoptotic protein Bcl-2. Over expression of thesegenes causes uncontrolled proliferation and survival of malignant cells,making knockdown of these genes by RNA interference (RNAi) a rationalestrategy for therapeutic intervention. RNAi is a conserved endogenousmechanism in which small interfering RNAs (siRNAs) suppresstarget-specific gene expression by promoting mRNA degradation. To targettwo of the critical B-cell lymphoma oncogenes, Bcl6 and STAT3, at thesame time, we have designed bifunctional siRNA duplexes using differentcomputer algorithms to predict accessible target sites in the mRNAs ofthe targets (Hossbach et al., 2006; http colon slash slash www dotmpibpc dot mpg dot de slash groups slash luehrmann slash siRNA). Thesebifunctional siRNAs contain two fully target-complimentary antisensestrands against Bcl6 and STAT3 mRNAs, respectively, but that are onlypartially complementary to each other. Bifunctional siRNAs have theadvantage to provide two antisense strands simultaneously suggesting thereduction of effective concentration transfected to the cells.Additionally, bifunctional siRNAs might also show increased specificityand decreased off-target effects compared to conventional 21mers due tothe lack of undesired activity of the passenger strand.

Different designs of the bifunctional siRNAs are possible: a) 21mer with2nt 3′ overhang at both strands (conventional siRNA design), b) 27merDicer-substrate with single 2nt 3′ overhang and 3′ DNA residues at theblunt end (asymmetrical design), c) 27mer with 2nt 3′ overhang at bothstrands, d) 27mer with blunt ends at both strand. All siRNAs werechemically synthesized. The structures of the bifunctional siRNAs areset forth below. Numbers indicate the target site in the mRNA sequencesof Bcl6 and STAT3, respectively. RNA bases are upper case, DNA bases arelower case. In vitro cleavage assays indicate that our bifunctionalsiRNAs have sufficient complementarity to form stable duplexes and canbe processed into smaller molecules by recombinant Dicer.

BS-1100 (Bcl6 1100, STAT3 3822)

BS-2755 (Bcl6 2755, STAT3 4576)

BS_1248 (Bcl6 1248, STAT3 1028)

To determine duplex formation and stability of bifunctional siRNAstargeting Bcl6 and STAT3, the sense and antisense strand of differentdesigns were denatured at 80° C. and then slowly cooled down to roomtemperature. The duplexes were analysed on a 15% native gel and stainedwith Sybr Gold (FIG. 12). A full complementary conventional siRNAagainst Bcl6 was used as reference (lane: Bcl6). All bifunctional siRNAsform duplexes, but most of the analyzed solutions contain single strandsbesides the duplex. Exceptions are BS-1100-27 and BS-1100-o that seem toform only stable duplexes.

Silencing efficacy was determined by psicheck assay (FIG. 13). Targetsites of the different siRNAs were cloned in the psicheck vector andcotransfected with corresponded bifunctional siRNA in HEK293 cells.Renilla and firefly luciferase activity was measured 24 h aftertransfection and values were normalized to the control. FIG. 13 showsclearly that the bifunctional siRNA BS-1100 is working against bothtargets.

To determine the efficacy of the bifunctional siRNAs, conventional21mers and asymmetrical 27mers were transfected into HEK293 cells usingcationic lipids (RNAiMax, Invitrogen) and total RNA was collected 48 hpost-transfection for quantitative RT-PCR. Target gene expression wasnormalized to levels of RPLP0 mRNA, a ribosomal protein that is anestablished reference gene. All analyzed bifunctional siRNAs were ableto reduce STATS mRNA levels by 50 to 80% as determined by quantitativeRT-PCR (FIG. 14).

The effect of the bifunctional siRNAs on interferon pathway relatedgenes in different cell types was analyzed using real-time PCR. Thereal-time PCR was performed for IFNβ, p56 and OAS1 mRNAs extracted fromRaji cells 48 h after electroporation of 2 μg siRNA following the Amaxaprotocol. As positive control, Raji cells were incubated with 1 U/μ1IFNα for 4 h. Data was normalized to RPLP0 and is shown in FIG. 15relative to the mock transfected control. Experiments were done induplicate. Similar results were obtained for Daudi, Su-DHL-4 andSu-DHL-6 cell lines. The results demonstrate that none of the analyzedsiRNAs show a significant increase in the expression of interferonpathway related genes in different cell lines.

In these experiments, we found that the most effective siRNAs reducetarget mRNA levels by ˜80% as determined by quantitative RT-PCR andimmunoblot analysis. The bifunctional siRNAs are able to silenceeffectively two critical B-cell lymphoma oncogenes at the same time. Inaddition, we found that the silencing of Bcl6 affects the expression ofdownstream target genes. Finally, we found that none of the analyzedsiRNAs show a significant increase in the expression of interferonpathway related genes in different cell lines.

Example 4 Targeting Two Genes on Separate Strands of siRNA

Multi-functional siRNAs are designed so that each strand of the duplextargets a specific sequence. In this design both strands of the siRNAduplex are used as triggers and can incorporate into various RISCcomplexes (FIG. 16A). The two strands of the designed siRNA duplex cantarget one mRNA at the 3′UTR and another mRNA at the coding region.Using HIV as an example, multi-strand siRNAs are designed so that onestrand targets the HIV 3′UTR and the other strand targets the HIV or theCCR5 coding region simultaneously. In our design the top strand, whichis the U2 anti-sense strand, functions as an miRNA against the HIV 3′UTRand the bottom strand, which has perfect complementarity against eitherthe CCR5 or the HIV coding region (Tables 4 and 5), functions as ansiRNA (FIG. 16B).

TABLE 4  Sequences of the Bottom Strand (siRNA strand) andTheir Mismatches with the U2 Anti-sense Strand (miRNA Strand) Mis-Target site (sense) matches ID Reporter (SEQ ID NO) w/ U2 U2-CCR5-3pCCR5 3′ AUCCCUAGUCUUCAAGCAG 7 UTR (41) U2-CCR5- CCR5 CDSUUUUCCAGCAAGAGGCUCCC 7 cds (42) U2-PolGag- pNL4-3  CCCACCAGAAGAGAGCUUCA9 cds PolGag (43) CDS U2-HIV1-3p HIV1 3′ ACACCAGGGCCAGGGGUCAG 11 UTR(44)

TABLE 5 Dicer Substrate Multi-strand siRNAs top-25 mer based on U2bottom-27 mer cleavage strand ID (SEQ ID NO) (SEQ ID NO) U2-CCR5-3puuccccagucaaaaguccaAUUGGA (45) uccaaucugcuugaagacuagggauuc (46)U2-CCR5-cds uuccccagucaaaaguccaCGAGCG (47)cgcucgggagccucuugcuggaagaua (48) U2-PolGag-cdsuuccccagucaaaaguccaAGGUUU (49) aaaccugaagcucucuucugguggggcu (50)U2-HIV1-3p uuccccagucaaaaguccaAUAUCC (51)ggauaucugaccccuggcccuggugugu (52) The top strand is the U2 anti-sensestrand (lower case) extended with nucleotides 3′ of the cleavage site(upper case). The bottom strand is reverse-complementary to the cleavagesite.

The predicted structures of the multi-functional siRNAs shown in Table 5are as follows.

U2-CCR5-3p:  (top: SEQ ID NO: 45; bottom: SEQ ID NO: 46): top    5′  U    C    AA     CCA       3′              UCCC AGUC   AAGU   AUUGGA             AGGG UCAG   UUCG   UAACCU bottom 3′CUU    A    AAG    UC        5′ U2-CCR5-cds: (top: SEQ ID NO: 47; bottom: SEQ ID NO: 48) top    5′     C    U   A       A       3′             UUC CCAG CAA AG  UCC CGAGCG            AAG GGUC GUU UC  AGG GCUCGC bottom 3′AU   A        C  CG           5′ U2-PolGag-cds: (top: SEQ ID NO: 49; bottom: SEQ ID NO: 50) top    5′           UCAAA   CCA       3′             UUCC CCAG     AGU   AGGUUU            GGGG GGUC     UCG   UCCAAA bottom 3′UC    U    UUCUC   AAG       5′ U2-HIV1-3p: (top: SEQ ID NO: 51; bottom: SEQ ID NO: 52) top    5′    C         AAA   CA       3′                CCA  GUCA   GUC  AUAUCC               GGU  CGGU   CAG  UAUAGG bottom 3′UGUGU   CC    CCC   UC       5′

The predicted target site interactions for CCR5_5 are as follows.

position 385:  (target: SEQ ID NO: 53; miRNA: SEQ ID NO: 54) target 5′G       C   A        U 3′            GCCUGGG GGG CUGGGGAG           CGGACCC CUC GACCCCUU miRNA  3′         U              5′position 343:  (target: SEQ ID NO: 55; miRNA: SEQ ID NO: 56) target 5′U   ACAA    CUUUCC        C 3′            GCU    GGGA      GCUGGGGA           CGG    CCCU      CGACCCCU miRNA  3′    A       CU            U 5′

Results of these multi-functional siRNAs are shown in FIGS. 17-20. FIGS.17A and 17B show the structures of U2 miRNA bound to the two 3′ UTRsites of HIV. FIGS. 18A and 18B show a schematic of the reporter pU.FIGS. 19A and 19B show a schematic of the reporter pCC. As shown in FIG.20, the expression of Renilla Luciferase gene was reduced to 44.46percent relative to the control irrelevant (pU-IRR) siRNA cotransfectedwith the pU reporter as well as the perfectly matched U2 siRNA (topcenter of the chart) transfected with the pU reporter (pU-U2), 50.69percent. The downregulation of the Renilla expression is indicatingprocessing of the Dicer-substrate multifunctional siRNA andincorporation of the U2 miRNA strand into RISC. The efficiency andpotency of the U2 miRNA in the context of the multifunctionalDicer-substrate duplex (left to right, first bar, 44.46%) was evenbetter compared to the conventional 21 mer perfect siRNA duplex of U2(left to right, second bar, 50.69%). The incorporation of the bottomstrand of the Dicer-substrate multifunctional siRNA was validated bycotransfecting the multi-functional siRNA with the reporter psiCheckconstruct containing one kilobase pairs of the CCR5 coding region (pCC,target). The expression of the Renilla Luciferase gene was reduced to37.71 percent (pCC-U2-C-C-D) (Left to right forth bar) relative to theirrelevant control (pCC-IRR) (Left to right, last bar). Thedownregulation of the Renilla expression is indicating processing of theDicer-substrate multifunctional siRNA and incorporation of the anti-CCR5strand into RISC.

All together, the data shows that multifunctional siRNA designs made upof two functional strands are recognized by the intracellular proteinmachineries responsible for RNAi phenomena. These novel siRNAs areprocessed efficiently by the Dicer protein complex and subsequentlyincorporated into appropriate RISC.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

BIBLIOGRAPHY

Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell 116:281-297.

Bertrand, E. et al. (1997). The expression cassette determines thefunctional activity of ribozymes in mammalian cells by controlling theirintracellular localization. RNA 3:75-88.

Birmingham, A. et al. (2006). 3′ UTR seed matches, but not overallidentity, are associated with RNAi off-targets. Nat Methods 3:199-204.

Chalk, A. M. et al. (2004). Improved and automated prediction ofeffective siRNA. Biochem Biophys Res Commun 319:264-274.

Doench, J. G. et al. (2003). siRNAs can function as miRNAs. Genes Dev17:438-442.

Eckstein, F. (2000). Phosphorothioate oligodeoxynucleotides: what istheir origin and what is unique about them? Antisense Nucleic Acid DrugDev 10:117-21.

Filipowicz, W. et al. (2005). Post-transcriptional gene silencing bysiRNAs and miRNAs. Curr Opin Struct Biol 75:331-341.

Good, P. D. et al. (1998). Expression of small, therapeutic RNAs inhuman cell nuclei. Gene Ther 4:45-54.

Griffiths-Jones, S. et al. (2006). miRBase: microRNA sequences, targetsand gene nomenclature. Nucleic Acids Res, 34, D140-144.

Haele, B. S. et al. (2005). siRNA target site secondary structurepredictions using local stable substructures. Nucleic Acids Res33(3):e30.

Hannon, G. J. and Rossi, J. J. (2004). Unlocking the potential of thehuman genome with RNA interference. Nature 431:371-378.

Herdewijn, P. (2000). Heterocyclic modifications of oligonucleotides andantisense technology. Antisense Nucleic Acid Drug Dev 10:297-310.

Hossbach, M. et al. (2006). Gene silencing with siRNA duplexes composedof target-mRNA-complementary and partially palindromic or partiallycomplementary single-stranded siRNAs. RNA Biol 3:82-89.

Kim, V. N. (2005). Small RNAs: classification, biogenesis, and function.Mol Cells 79:1-15.

Kreuter, J. (1991). Nanoparticles-preparation and applications. In:Microcapsules and nanoparticles in medicine and pharmacy, Donbrow M.,ed, CRC Press, Boca Raton, Fla., pp. 125-14.

Lim, L. P. et al. (2005). Microarray analysis shows that some microRNAsdownregulate large numbers of target mRNAs. Nature 433:769-773.

Liu, J. et al. (2005). MicroRNA-dependent localization of targeted mRNAsto mammalian P-bodies. Nat Cell Biol 7:719-723.

Paddison, P. J. et al. (2002). Short hairpin RNAs (shRNAs) inducesequence-specific silencing in mammalian cells. Genes Dev 16:948-958.

Rajewsky, N. (2006). microRNA target predictions in animals. Nat Genet38 Suppl:S8-13.

Rusckowski, M. et al. (2000). Biodistribution and metabolism of a mixedbackbone oligonucleotide (GEM 231) following single and multiple doseadministration in mice. Antisense Nucleic Acid Drug Dev 10:333-345.

Saetrom, P. (2004). Predicting the efficacy of short oligonucleotides inantisense and RNAi experiments with boosted genetic programming.Bioinformatics 20:3055-3063.

Saetrom, P. and Snove, O., Jr. (2004). A comparison of siRNA efficacypredictors. Biochem Biophys Res Commun 321:247-253.

Saetrom, P. et al. (2007). Manuscript submitted.

Snove, O. and Rossi, J. J. (2006). Expressing short hairpin RNAs invivo. Nature Methods 3:689-695.

Stein, D. A. et al. (2001) Inhibition of Vesivirus infections inmammalian tissue culture with antisense morpholino oligomers. AntisenseNucleic Acid Drug Dev 11:317-25.

Verma, S. And Eckstein, F. (1998). Modified oligonucleotides: synthesisand strategy for users. Annu Rev Biochem 67:99-134.

Vert, J. P. et al. (2006). An accurate and interpretable model for siRNAefficacy prediction. BMC Bioinformatics 7:520 (17 pages).

Vorobjev, P. E. et al. (2001). Nuclease resistance and RNase Hsensitivity of oligonucleotides bridged by oligomethylenediol andoligoethylene glycol linkers. Antisense Nucleic Acid Drug Dev 11:77-85.

Wu, L. et al. (2006). MicroRNAs direct rapid deadenylation of mRNA. ProcNatl Acad Sci USA 103:4034-4039.

Zeng, Y. et al. (2002). Both natural and designed micro RNAs can inhibitthe expression of cognate mRNAs when expressed in human cells. Mol Cell9:1327-1333.

What is claimed is:
 1. A method of treating HIV-1 infection which comprises administering a therapeutically effective amount of a multi-targeting RNA molecule to an individual in need thereof, wherein the multi-targeting RNA molecule targets two or more different desired target sequences, wherein the RNA molecule comprises a first strand having a 5′ terminus and a 3′ terminus and a second strand having a 5′ terminus and a 3′ terminus, wherein the first strand is perfectly complementary to a desired mRNA target sequence, wherein the second strand contains two or more seed matches optimally spaced in a desired 3′ UTR containing target gene, wherein the first strand and second strand are not completely complementary and wherein the first strand and the second strand hybridize to form a double stranded RNA molecule, wherein the double stranded RNA molecule is a single duplex, wherein the first strand and the second strand have nucleotide sequences selected from the following group: (a) first strand: (SEQ ID NO: 46) 5′ uccaaucugcuugaagacuagggauuc 3′ and second strand: (SEQ ID NO: 45) 5′ uuccccagucaaaaguccaauugga 3′; (b) first strand: (SEQ ID NO: 48) 5′ cgcucgggagccucuugcuggaagaua 3′ and second strand: (SEQ ID NO: 47) 5′ uuccccagucaaaaguccacgagctg 3′; (c) first strand: (SEQ ID NO: 50) 5′ aaaccugaagcucucuucugguggggcu 3′ and second strand:  (SEQ ID NO: 49) 5′ uuccccagucaaaaguccaagguuu 3′; and (d) first strand: (SEQ ID NO: 52) 5′ ggauaucugaccccuggcccuggugugu 3′ and second strand:  (SEQ ID NO: 51) 5′ uuccccagucaaaaguccaauaucc 3′.


2. The method of claim 1, wherein the optimal spacing between seed matches is between 13 and 100 nucleotides.
 3. The method of claim 2, wherein the optimal spacing between seed matches is between 13 and 35 nucleotides.
 4. The method of claim 1, wherein the first strand results in cleavage of the desired mRNA target sequence and wherein the seed matches result in down-regulation of the desired 3′ UTR containing target gene.
 5. The method of claim 4, wherein the desired mRNA target sequence and the desired 3′ UTR containing target gene are the same gene.
 6. The method of claim 5, wherein the gene is an HIV gene.
 7. The method of claim 4, wherein the desired mRNA target sequence is a target sequence of a first desired gene and the desired 3′ UTR containing target gene is a second desired gene.
 8. The method of claim 7, wherein the first desired gene is CCR5 and the second desired gene is 3′ UTR of HIV. 